Cloranfenicol Succinato de Sodio

(Ph. Eur. monograph 0709)

C15H15Cl2N2NaO8  445.2  982-57-0

Action and use

Antibacterial.

Preparation

Chloramphenicol Sodium Succinate Injection

Ph Eur

DEFINITION

Mixture in variable proportions of sodium (2R,3R)-2-[(dichloroacetyl)amino]-3- hydroxy-3-(4-

nitrophenyl)propyl butanedioate (3 isomer) and of sodium (1R,2R)-2-[ (dichloroacetyl)amino]-3-
hydroxy-1-(4-nitrophenyl)propyl butanedioate (1 isomer).

Semi-synthetic product derived from a fermentation product.

Content

98.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS

Appearance

White or yellowish-white powder, hygroscopic.

Solubility

Very soluble in water, freely soluble in ethanol (96 per cent).

IDENTIFICATION

 A. Thin-layer chromatography (2.2.27).

Test solution Dissolve 20 mg of the substance to be examined in 2 mL of acetone R.

Reference solution (a) Dissolve 20 mg of chloramphenicol sodium succinate CRS in 2 mL of

acetone R.

Reference solution (b) Dissolve 20 mg of chloramphenicol CRS in 2 mL of acetone R.

Plate TLC silica gel GF254 plate R.

Mobile phase dilute acetic acid R, methanol R, chloroform R (1:14:85 V/V/V).

Application 2 µL.

Development Over a path of 15 cm.

Drying In air.

Detection Examine in ultraviolet light at 254 nm.

Results The 2 principal spots in the chromatogram obtained with the test solution are similar in
position and size to the 2 principal spots in the chromatogram obtained with reference solution (a);
their positions are different from that of the principal spot in the chromatogram obtained with
reference solution (b).

 B. Dissolve about 10 mg in 1 mL of ethanol (50 per cent V/V) R, add 3 mL of a 10 g/L solution of
calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot
solution and allow to cool. Add 0.1 mL of benzoyl chloride R and shake for 1 min. Add 0.5 mL of
ferric chloride solution R1 and 2 mL of chloroform R and shake. The upper layer is light violet-red
or purple.

 C. Dissolve 50 mg in 1 mL of pyridine R. Add 0.5 mL of dilute sodium hydroxide solution R and
1.5 mL of water R. Heat in a water-bath for 3 min. A red colour develops. Add 2 mL of nitric acid R
and cool under running water. Add 1 mL of 0.1 M silver nitrate. A white precipitate is formed slowly.

 D. It gives reaction (a) of sodium (2.3.1).

TESTS

pH (2.2.3)

6.4 to 7.0.

Dissolve 2.50 g in carbon dioxide-free water R and dilute to 10 mL with the same solvent.

Specific optical rotation (2.2.7)

+ 5.0 to + 8.0 (anhydrous substance).

Dissolve 0.50 g in water R and dilute to 10.0 mL with the same solvent.

Chloramphenicol and chloramphenicol disodium disuccinate

Liquid chromatography (2.2.29).

Test solution Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to
100.0 mL with the mobile phase.

Reference solution (a) Dissolve 10.0 mg of chloramphenicol CRS in the mobile phase and dilute
to 100.0 mL with the mobile phase (solution A). Dilute 5.0 mL of this solution to 100.0 mL with the
mobile phase.

Reference solution (b) Dissolve 10.0 mg of chloramphenicol disodium disuccinate CRS in the

mobile phase and dilute to 100.0 mL with the mobile phase (solution B). Dilute 5.0 mL of this
solution to 100.0 mL with the mobile phase.

Reference solution (c) Dissolve 25 mg of the substance to be examined in the mobile phase, add
5 mL of solution A and 5 mL of solution B and dilute to 100 mL with the mobile phase.

Column:
 — size: l = 0.25 m, Ø = 4.6 mm;

 — stationary phase: octadecylsilyl silica gel for chromatography R (5 µm).

Mobile phase 20 g/L solution of phosphoric acid R, methanol R, water R (5:40:55 V/V/V).

Flow rate 1.0 mL/min.

Detection Spectrophotometer at 275 nm.

Injection 20 µL.

System suitability Reference solution (c):

 — the 2 peaks corresponding to those in the chromatograms obtained with reference solutions
(a) and (b) are clearly separated from the peaks corresponding to the 2 principal peaks in the
chromatogram obtained with the test solution; if necessary, adjust the methanol content of the
mobile phase.

Limits:
 — chloramphenicol: not more than the area of the principal peak in the chromatogram obtained
with reference solution (a) (2.0 per cent);

 — chloramphenicol disodium disuccinate: not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (2.0 per cent).

Water (2.5.12)

Maximum 2.0 per cent, determined on 0.500 g.

Pyrogens (2.6.8)
If intended for use in the manufacture of parenteral preparations without a further appropriate
procedure for removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the
rabbit's mass 2.5 mL of a solution in water for injections R containing 2 mg of the substance to be
examined per millilitre.

ASSAY

Dissolve 0.200 g in water R and dilute to 500.0 mL with the same solvent. Dilute 5.0 mL of this
solution to 100.0 mL with water R. Measure the absorbance (2.2.25) at the absorption maximum at
276 nm.

Calculate the content of C15H15Cl2N2NaO8, taking the specific absorbance to be 220.

STORAGE

In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight,
tamper-proof container, protected from light.

Cloranfenicol (materia prima)

(Ph. Eur. monograph 0071)

C11H12Cl2N2O5  323.1  56-75-7

Action and use

Antibacterial.

Preparations

Chloramphenicol Capsules

Chloramphenicol Ear Drops

Chloramphenicol Eye Drops

Chloramphenicol Eye Ointment

Ph Eur

DEFINITION

Chloramphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4-
nitrophenyl)ethyl]acetamide, produced by the growth of certain strains of Streptomyces venezuelae
in a suitable medium. It is normally prepared by synthesis. It contains not less than 98.0 per cent
and not more than the equivalent of 102.0 per cent of C 11H12Cl2N2O5, calculated with reference to
the dried substance.

CHARACTERS

A white, greyish-white or yellowish-white, fine, crystalline powder or fine crystals, needles or

elongated plates, slightly soluble in water, freely soluble in alcohol and in propylene glycol.

A solution in ethanol is dextrorotatory and a solution in ethyl acetate is laevorotatory.

IDENTIFICATION

First identification A, B.

Second identification A, C, D, E.

 A. Melting point (2.2.14): 149 °C to 153 °C.

 B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with chloramphenicol CRS.

 C. Examine the chromatograms obtained in the test for related substances. The principal spot in
the chromatogram obtained with 1 µL of the test solution is similar in position and size to the
principal spot in the chromatogram obtained with reference solution (a).

 D. Dissolve about 10 mg in 1 mL of alcohol (50 per cent V/V) R, add 3 mL of a 10 g/L solution of
calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot
solution and allow to cool. Add 0.1 mL of benzoyl chloride R and shake for 1 min. Add 0.5 mL of
ferric chloride solution R1 and 2 mL of chloroform R and shake. The aqueous layer is coloured light
violet-red to purple.
 E. To 50 mg in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an
open flame for 10 min. Allow to cool. Take up the residue with 5 mL of dilute nitric acid R and filter.
To 1 mL of the filtrate add 1 mL of water R. The solution gives reaction (a) of chlorides (2.3.1).

TESTS

Acidity or alkalinity

To 0.1 g add 20 mL of carbon dioxide-free water R, shake and add 0.1 mL of bromothymol blue
solution R1. Not more than 0.1 mL of 0.02 M hydrochloric acid or 0.02 M sodium hydroxide is
required to change the colour of the indicator.

Specific optical rotation (2.2.7)

Dissolve 1.50 g in ethanol R and dilute to 25.0 mL with the same solvent. The specific optical
rotation is + 18.5 to + 20.5.

Related substances

Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.

Test solution Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10 mL

with the same solvent.

Reference solution (a) Dissolve 0.10 g of chloramphenicol CRS in acetone R and dilute to 10 mL

with the same solvent.

Reference solution (b) Dilute 0.5 mL of reference solution (a) to 100 mL with acetone R.

Apply separately to the plate 1 µL and 20 µL of the test solution, 1 µL of reference solution (a) and
20 µL of reference solution (b). Develop over a path of 15 cm using a mixture of 1 volume of water
R, 10 volumes of methanol R and 90 volumes of chloroform R. Allow the plate to dry in air and
examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with 20 µL of the test
solution, apart from the principal spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent).

Chlorides (2.4.4)

To 1.00 g add 20 mL of water R and 10 mL of nitric acid R and shake for 5 min. Filter through a
filter paper previously washed by filtering 5 mL portions of water R until 5 mL of filtrate no longer
becomes opalescent on addition of 0.1 mL of nitric acid R and 0.1 mL of silver nitrate solution R1.
15 mL of the filtrate complies with the limit test for chlorides (100 ppm).

Loss on drying (2.2.32)

Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C.

Sulfated ash (2.4.14)

Not more than 0.1 per cent, determined on 2.0 g.

Pyrogens (2.6.8)
 If intended for use in the manufacture of parenteral preparations without a further appropriate
procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of
the rabbit's mass 2.5 mL of a solution containing per millilitre 2 mg of the substance to be
examined.

ASSAY

Dissolve 0.100 g in water R and dilute to 500.0 mL with the same solvent. Dilute 10.0 mL of this
solution to 100.0 mL with water R. Measure the absorbance (2.2.25) at the maximum at 278 nm.

Calculate the content of C11H12Cl2N2O5 taking the specific absorbance to be 297.

STORAGE

Store protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof
container.